Injectable solution with anti-inflammatory effect and process for manufacturing the same

ABSTRACT

The present invention relates to an injectable solution with anti-inflammatory effect which has in its composition 1 . . . 700 mg active powder containing water soluble inorganic salts, obtained from the ash resulted by Symphytum officinale roots calcination, in 100 ml distilled water or saline solution, optionally associated with 0.1-0.2 mg Lidocaine for the intramuscular administrating injectable solution.

[0001] The present invention relates to an injectable solution, withanti-inflammatory effect, a process for manufacturing the same and amethod for the treatment of autoimmune diseases using the said solution.

[0002] It is known that an increased number of human diseases, e.g.nephrosis, rheumatoid arthritis, dermatomyositis, chronic hepatitis,leukocytoclastic vasculitis, etc., may be designated as autoimmunedisorders. It is generally acknowledged that pathophysiology ofimmune-complexes, i.e. an autoimmune response following viral, bacterialor parasitic aggression leads to qualitative and quantitativealterations in leukocytes named lymphocytes which are involved in theorganism immune defense. An important role in restoring the immuneresponse was proved to be played by drugs with immuno-modulatingactivity, which, thanks to their composition, rehabilitate the saltbalance of the internal human body medium, stimulate the lymphocytedivision, promote antibody synthesis and enhance the phagocytic capacity(Franco Dammaco, “Imunologia in Medicina”, Edi. Ermes, Milano, 1989, p67 cap 3.15, tab. 3.2, p 98 tab. 19.8, p 679).

[0003] Various drugs, which are obtained from medicinal herbs, withactive principles having therapeutical qualities for the treatment ofimmunodeficiency syndrome, are known in the art. Such a product is acomposition obtained in the form of a vegetal extract derived from theair segments of the Chimaphila umbellate, Apocynum androsaemifolium,Symphytum officinale and Equisetum hyemale (WO 97-04793).

[0004] The roots of Symphytum officinale are known to be used asexternal application for the treatment of hard healing wounds, provingthat this plant segment has real hemostatic and healing capacities.These properties have been attributed to the allantoin content(0.6-0.8%), a toxicity-free principle (Evdochia Coiciu and Gabriel Racz,“Plante medicinale

i aromatice”, 1962, p 550-551). Due to these properties, the Symphytumofficinale plant has also been used in pharmaceutical industry where,through different extraction processes, the active principles obtainedhave been used in various pharmaceutical forms, especially for externalapplication, e. g. emulsions for the treatment of skin disorders. Theobject of the extraction processes is mainly the extraction of allantoinfrom a polyphase fluid (WO 92-06701 and EP 0673654 A1).

[0005] It comes out that, until now, Symphytum officinale plant has beenespecially exploited for the extraction of its organic activeprinciples, e. g. allantoin and mucilage contained without anyinvestigation of the therapeutical properties of its inorganicprinciples.

[0006] The technical matter the present invention solves is exactly theresearch achievement and ascertaining of the therapeutical effects ofthe inorganic constituents of the Symphytum officinale plant and thepreparation of a drug in the form of an injectable solution to confirmthe real anti-inflammatory properties this plant possesses also throughits inorganic constituents.

[0007] The injectable solution according to the invention comprises 1 .. . 700 mg active powder containing water soluble inorganic saltsobtained from the ash resulted by the calcination of Symphytumofficinale roots, in 100 ml distilled water or saline solution,optionally associated with 0.1-0.2 mg Lidocaine for the intramuscularadministrating injectable solution.

[0008] The active powder of inorganic salts termed IMUNOMOD containspotassium salts in the range of 36 to 52% and, stoichiometrically,comprises 43.268% potassium, 14.698% sulphur, 31.675% oxygen, 0.88%iron, 0.421% sodium, 0.265% phosphorus, 0.126 chlorine, 0.043% silicon,0.034% magnesium, 0.012% manganese and cassiopeum traces, and up to 100%being carbon from carbonates and organic residues, e. g. alkali metaloxalates.

[0009] The process for manufacturing the injectable solution consists ofthe following steps:

[0010] a) the cutting of the Symphytum officinale roots and the dryingof the vegetal material;

[0011] b) the calcination of the dried vegetal material at a temperatureof 550-600° C. in calcination containers known per se, over a period of3-4 hours;

[0012] c) the ash obtained after calcination is dissolved in distilledwater at a ratio of 1:100, the obtained solution is filtered undervacuum, followed by filtration through milipor or sympor filters and thefiltrate is subjected to boiling evaporation up to a viscousness of6.57±0.5 cP;

[0013] d) the viscid liquid is placed in thin layer on Pyrex glass pansand is subjected to drying at a temperature of 200-250° C., over aperiod of 5-15 minutes until a white powder is obtained;

[0014] e) for preparing the injectable solution, the powder is dissolvedin distilled water in amount of 1 . . . 700 mg to 100 ml distilled wateror saline solution, optionally associated with 0.1 mg Lidocaine for theintramuscular injectable solution.

[0015] The method for the treatment of autoimmune diseases according tothe invention involves a three step regimen administration of theintramuscular injectable solution, i. e. in the first step, the dailyadministration of a dose of minimum 30 mg/70 kg body weight, over aperiod of 10 consecutive days, then, in the second step, theadministration is performed with the same dose, at free intervals of 1to 2 days, until obvious positive clinical results are obtained,afterwards one proceeds to the maintenance step of the treatment by oncea week intramuscular administration, in the same dose, of the injectablesolution, alternating with intravenous administration.

[0016] The medical product according to the invention has the followingadvantages:

[0017] possesses anti-inflammatory activity proved by the dynamics of a2-fraction in the blood electrophoresis, its value decreasing relatedwith the inflammatory effect decline respectively;

[0018] produces no allergic reaction proved by quantitative levellowering of eosinophyles established by blood cell count, and thus thereis no need for previous testing, but for the injectable form containingLidocaine (only for the Lidocaine allergical test);

[0019] acts as a MCSF factor (Multi Colony Stimulating Factor)reestablishing to normal the levels of serum leukocytes, erithrocytesand platelets;

[0020] its manufacturing process is very simple and reproducible forobtaining its therapeutical qualities.

[0021] Two Examples for manufacturing the injectable solution accordingto the invention as well as the method for treatment are presentedfurther on.

EXAMPLE 1

[0022] The fresh harvested roots of Symphytum officinale are washedunder water stream and subjected to cutting in pieces of about 4-5 cm.The vegetal product obtained is subjected to drying following normsknown per se for the conservation of the medicinal plant roots.

[0023] 1000 g cut and dried roots of Symphytum officinale are subjectedto calcination in a temperature controlled furnace in therm-resistantporcelain containers at a temperature of 550-600° C., over a period of34 hours. 250-300 g light grey ash are yield. The ash thus obtained isdissolved in distilled water at a ratio of 1:100, at room temperature,with stirring over a period of 15 minutes, in glass containers. Theliquid obtained is filtered under vacuum using a Nuce filter, forremoving the undissolved particles. The resulted solution is subjectedagain to filtration using Milipor and Sympor filters. The filtrateobtained has the following ion composition: K⁺=36.25-44.48%;Na⁺=1.89-2.70%; Cl⁻=0.18-0.36%; HCO₃ ⁻=34.12-55.3%; CO₃ ²⁻=14.71-18.9%and SO₄ ²⁻=0.96-1.20%.

[0024] IR analyses indicated vibrations corresponding mainly to HCO₃ ⁻,CO₃ ²⁻, OH⁻ anions and hydrogen bonds.

[0025] The obtained filtrate is subjected to boiling evaporation inPyrex glass recipients up to a liquid viscosity of 6.57±0.5 cP. Thusobtained viscid liquid is placed on Pyrex pans in thin layer andsubjected to drying at 200-250° C., over a period of 5-10 minutes, thusobtaining an easy adhesive white powder. Right after the drying, thepowder is removed using stainless tools, known per se, and introduced intight ampules because of its high hygroscopic potential.

[0026] There are obtained about 40-50 g active powder called IMUNOMODwhich in IR presents vibrations at 3400, 1630, 1110, 1004, 880, 845,700, 686, 672, 640, 625, 618 and 560, and by X-ray diffraction shows thepresence of KHCO₃, K₂CO₃, KCl, K₂SO₄ and C₄H₃KO₈.2H₂O (potassiumoxalates hydrates) crystals. These conclusions are shown by the electronmicroscopic image and by the IR spectrum chart.

[0027] The stoichiometrical analyses at the electron microprobe showsthe following chemical composition: K=43.268%; S=14.698%; O=31.675%;Fe=0.88%; Na=0.421%; P=0.265%; Cl=0.126%; Si=0.045%; Mg=0.034%;Mn=0.012% and up to 100% being carbon within carbonates and organicresidues, such as the alkali metal oxalates.

[0028] As a result of the X-ray microanalysis, on the JCXA-50A electronmicroprobe, provided with AN 10000 X-ray analyzer (lithium doped silicontype detector, cooled in a cryogenic room, at the liquid nitrogentemperature), all the elements, heavier than sodium (Na) have beendetected in the IMUNOMOD active powder, and among these, cassiopeum wasdetected too.

[0029] The radioactivity analysis of the active powder established thatit is under the admitted limit.

[0030] The thermogravimetric analysis of the IMUNOMOD active powderindicated weight losses (by decomposition) on the following temperatureranges:

[0031] loss of 2 . . . 4% from the powder weight up to 100° C.;

[0032] loss of 13 . . . 14% from the powder weight up to 185° C.;

[0033] loss of 17% from the powder weight up to 280° C.;

[0034] loss of 20% from the powder weight up to 800° C.

[0035] All the decomposition effects being endothermal, indicatingdecomposition processes, and almost the lack of exothermal processes(burnings), indicates a low percentage of organic compounds.

[0036] IMUNOMOD active powder is formulated as a solution by itsdissolving in distilled water, in amount of 600 mg to 100 ml distilledwater, with an addition of 10 ml 1% Lidocaine solution. This solution isdesignated to fit in 5 ml ampules for intramuscular administration andthe powder/ampoule concentration is given by the minimal dose for anadult/day/70 kg body weight, so the minimum daily dose would be one 5 mlampoule, containing 30 mg active powder.

[0037] For intravenous administration, IMUNOMOD active powder isdissolved in saline solution, in amount of 200 mg to 100 ml salinesolution without Lidocaine addition. This solution is designated to fitin 15 ml ampules for intravenous administration, the activepowder/ampoule concentration is also given by the minimal dose for anadult/day/70 kg body weight, i.e. 30 mg active powder in 15 mlintravenous injectable solution.

[0038] Thus obtained solutions are fitted in unidose ampules, in asepticmedium, and are subjected to sterilization according to RomanianPharmacopeia, X^(th) edition.

[0039] The injectable solution for adults, designated for intramuscularadministration, has to contain 30 mg active powder as a unidose (a 5 mlampoule contains 30 mg active powder).

[0040] The injectable solution for adults, designated for intramuscularadministration, has to contain also 30 mg active powder as a unidose (a15 ml ampoule contains 30 mg active powder).

[0041] The method for the treatment of chronic inflammatory diseaseswith injectable solution, according to the invention, presumesintramuscular and intravenous administration of the product concordantwith a regimen of the following steps: in the first step, the dailyadministration, over a period of 10 consecutive days, then, in thesecond step, the administration is performed with free intervals of 1-2days, until obvious positive clinical results are obtain, afterwards oneproceeds to the maintenance step of the treatment by once a weekintramuscular administration, alternating with intravenousadministration of the injectable solution.

[0042] The minimal dose for intramuscular administration to an adult isof 30 mg/day/70 kg body weight, i.e. the administration of one 5 mlampoule.

[0043] For intravenous administration, the minimal dose is also of 30mg/day/70 kg body weight, i.e. the administration of one 15 ml ampoule.Note that intravenous administration of the injectable solutionaccording to the invention is performed very slowly, over a period ofminimum 10 minutes, thanks to a very good tolerance of the injectablesolution to slow administration.

[0044] The method of treatment may be applied for chronic autoimmuneinflammatory diseases, such as nephrosis, rheumatoid arthritis,dermatomyositis, leukocytoclastic vasculitis, myasthenia gravis,ulcer-hemorrhagic recto colitis, autoimmune chronic hepatitis.

[0045] In the case of nephrosis, the injectable solution administration,according to the invention, is initially performed simultaneously withcorticosteroid therapy, at least over a period of 10 days during thetime the injectable solution according to the invention is administrateddaily. In the second step of the method for treatment according to theinvention, as soon as the positive effects occur (the edema retractionand the improvement of the α2-fraction level in electrophoresis), a slowdecrease of the daily cortisone dose will be started, up to the totalcortisone excretion over a period of about 3-4 months, during which theinjectable solution is administrated weekly. At the end of the 4 monthsof treatment a notable edema retraction, along with the body weightloss, and electrophoresis correction are noticed.

[0046] In the case of arthritis, carrying out the method for treatmentaccording to the invention, leads to joint pain relieve, decreasing andnormalizing the values of the following analyses: ESR, reactive Cprotein, rheumatoid factor, cryoglobulines, ASLO, fibrinogen.

[0047] In the case of dermatomyositis, carrying out the method accordingto the invention leads to complete rebuilding of the muscular system,alleviating the muscular, nervous and joint discomfort, extremedecreasing or withdrawal of corticosteroid therapy. In the same time,ASLO will be restored to normal values.

[0048] In the case of leukocytoclastic vasculitis, carrying out themethod according to the invention leads to joint pain relieve,erythematous areas and related pains diminishing. Regarding thelaboratory analyses, erythrocyte sedimentation rate and high fibrinogenlevel decreasing is noticed.

[0049] In the case of myasthenia gravis, carrying out the methodaccording to the invention results in alleviating the phonation,mastication or deglutition difficulties and diplopia disappearance.

[0050] In the case of ulcer-hemorrhagic recto colitis, when the methodaccording to the invention is applied, a normalizing of stool aspect,i.e. stool without blood, consistent, solid and colored, is detected.

[0051] In the case of chronic autoimmune hepatitis, applying the methodaccording to the invention results in the normalizing of liverultrasound aspect from nodular to normal liver, and from oversized tonormally sized. Regarding the laboratory analyses, the following returnto normal: transaminases, bilirubin, and electrophoresis.

[0052] Applying the method according to the invention is an importantadjuvant for the AIDS treatment, through its immunity increasing andopportunistic infection induced inflammatory effect decreasing.

EXAMPLE 2

[0053] The process for manufacturing the IMUNOMOD active powder from theSymphytum officinale roots is identical to the one described in Example1.

[0054] The ash obtained from the vegetal product calcination isdissolved in distilled water, in similar conditions to those in Example1, and the obtained filtrate has the same composition.

[0055] The obtained filtrate is subjected to boiling evaporation inPyrex glass recipients, up to a liquid viscousness of 6.57±0.5 cP. Theviscid liquid thus obtained is placed on Pyrex pans, in thin layer, andis dried at 200-250° C., over a period of 5-10 minutes, thus obtainingan easy adhesive white powder. Right after the drying, the powder isdetached using stainless tools known per se, and is introduced in tightflask, due to its great hygroscopic potential.

[0056] The IMUNOMOD active powder has the same composition as the one inExample 1.

[0057] In this Example for manufacturing the injectable solutionaccording to the invention, the solution is designated to theadministration in children with the same disorders as in adults.

[0058] For the injectable solution administration in 2-3 years oldchildren, the IMUNOMOD active powder is formulated as a solution by itsdissolving in distilled water, in amount of 150 mg to 100 ml distilledwater, with an addition of 20 ml 1% Lidocaine solution. This solution isdesignated to fit in 2 ml ampules for intramuscular administration, andpowder/ampoule concentration is given by the minimal dose for the 2-3years old child/day/12 kg body weight, so the minimal daily dose wouldbe a 2 ml ampoule, containing 7,5 mg active powder.

[0059] For the injectable solution administration, in 4-12 years oldchildren, the IMUNOMOD active powder is formulated as a solution, by itsdissolving in distilled water, in amount of 300 mg to 100 ml distilledwater, with an addition of 20 ml of 1% Lidocaine solution. This solutionis designated to fit in 2 ml ampules for intramuscular administration,and powder/ampoule concentration is given by the minimal dose for the4-12 years old child/day/20 kg body weight, so the minimal dose would bea 2 ml ampoule, containing 15 mg active powder.

[0060] For children over 12 years of age, the injectable solution hasthe same active powder concentration as in adult case, noting that theunidose will be calculated on the body weight.

[0061] Intravenous administration in children should be performed onlyfor those over 12 years of age, due to difficult manipulation of venousvessels, and to psychomotor stability increasing after this age. Inthese cases, the intravenous administration is also made slowly, over aperiod of 10 minutes. For the intravenous administration, the IMUNOMODactive powder is dissolved in saline solution, in amount of 100 mg to100 ml saline solution, without Lidocaine addition. This solution isdesignated to fit in 15 ml ampoules for intravenous administration,active powder/ampoule concentration is of 30 mg active powder in 15 mlintravenous injectable solution and the unidose is also given by thebody weight.

[0062] Thus obtained injectable and intramuscular administrationdesignated solutions are fitted in ampules assigned for unidoses (2 mlfor 2-12 years old children and 5 ml for children over 12 years of age)in aseptic medium are subjected to sterilization in the terms stipulatedin Romanian Pharmacopeia, X^(th) edition.

[0063] The injectable solutions for intravenous administration arefitted in 15 ml ampules as described in Example 1. Note that theintravenous administration of the injectable solution according to theinvention is performed very slowly, over a period of minimum 10 minutes.

[0064] The method for treatment in children, in the case of chronicinflammatory disease, with the injectable solution according to theinvention is similar to the one described in adult cases, noting thatthe intravenous administration is performed only in children over 12years of age.

[0065] In the case of children less than 2 years of age, the solutionaccording to the invention formulated at a concentration of 150 mg to100 ml distilled water (without Lidocaine addition) may be orallyadministrated at doses and by a method for treatment according to thepresent invention.

[0066] The method for treatment may be also applied in children withchronic autoimmune inflammatory diseases, e. g. nephrosis, rheumatoidarthritis, dermatomyositis, chronic autoimmune hepatitis.

[0067] In the case of nephrosis, the injectable solution administrationaccording to the invention is highly effective. The administration isinitially performed simultaneously with corticosteroid therapy, at leastover a period of 10 days during the time the injectable solutionaccording to the invention is administrated daily. In the second step ofthe method for treatment according to the invention, as soon as thepositive effects occur (the edema retraction and the improvement of theα2-fraction level in electrophoresis), a slow decrease of the dailycortisone dose will be started, until the total cortisone excretion,over a period of about 3-4 months, during which the injectable solutionis administrated weekly. At the end of the 4 months of treatment anotable edema retraction, along with the body weight loss, andelectrophoresis correction are noticed, and after 11 months ofmaintenance treatment without corticosteroid therapy, the Cushing aspectcompletely disappears, and the height gain is notable.

[0068] In the case of arthritis, applying in children the method fortreatment according to the invention results in joint tumefactionreduction, pain disappearance and normalizing the following analyses:ESR, reactive protein C, rheumatoid factor, cryoglobulines, ASLO,fibrinogen. The height gain is important and the visual deficit inducedby the associated iridocyclitis, is considerably remitted.

[0069] In the case of chronic autoimmune hepatitis, applying the methodaccording to the invention results in liver ultrasound aspectnormalizing, from nodular to normal liver aspect, and from oversized tonormally sized. Regarding the laboratory analyses, the following valuesreturn to normal: transaminases, bilirubin and electrophoresis.

1. Injectable solution with anti-inflammatory effect, characterized inthat it has in its composition 1 . . . 700 mg active powder containingwater soluble inorganic salts, obtained from the ash resulted bySymphytum officinale roots calcination, in 100 ml distilled water orsaline solution, optionally associated with 0.1-0.2 mg Lidocaine for theintramuscular administrating injectable solution.
 2. Injectable solutionwith anti-inflammatory effect according to claim 1, characterized inthat the active powder has in its composition 36-52% potassium saltsand, stoichiometrically, comprises 43.268% potassium, 14.698% sulphur,31.675% oxygen, 0.88% iron, 0.421% sodium, 0.265% phosphorus, 0.126chlorine, 0.043% silicon, 0.034% magnesium, 0.012% manganese andcassiopeum traces, and up to 100% being carbon from carbonates andorganic residues, e. g. alkali metal oxalate.
 3. Process formanufacturing the injectable solution, characterized in that presumesthe following steps: a) the cutting of the Symphytum officinale rootsand drying of the vegetal material; b) the calcination of the vegetalmaterial dried at a temperature of 550-600° C. in calcination containerknown per se, over a period of 3 to 4 hours; c) the ash obtained aftercalcination is dissolved in distilled water at a ratio of 1:100, theobtained solution is filtered under vacuum, followed by filtrationthrough milipor or semper filters and the filtrate is subjected toboiling evaporation up to a viscousness of 6.57±0.5 cP; d) the viscidliquid is placed in thin layer on Pyrex glass pans and is subjected todrying at a temperature of 200-250° C., over a period of 5-15 minutesuntil a white powder is obtained; e) for preparing the injectablesolution, the powder is dissolved in distilled water in amount of 1 . .. 700 mg to 100 ml distilled water or saline solution, optionallyassociated with 0.1 mg Lidocaine for the intramuscular injectablesolution.
 4. Method for the treatment of autoimmune diseases,characterized in that it presumes a three step regimen administration ofthe injectable solution, i. e. in the first step, the dailyintramuscular administration in an adult of a dose of minimum 30 mg/70kg body weight, over a period of 10 consecutive days, then, in thesecond step, the administration is performed with the same dose, at freeintervals of 1 to 2 days until obvious positive clinical results areobtained, afterwards one proceeds to the maintenance step of thetreatment by once a week intramuscular administration, in the same dose,of the injectable solution, alternating with intravenous administration.5. Method for the treatment of autoimmune diseases according to claim 4,characterized in that the injectable solution administration in childrenless than 12 years of age is made only by intramuscular route, in. dosesof minimum 7.5 mg/70 kg body weight.